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1.  Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R-loop accumulation 
Molecular cell  2014;53(3):484-497.
SUMMARY
Tudor domain containing protein 3 (TDRD3) is a major methyl-arginine effector molecule that “reads” methyl-histone marks and facilitates gene transcription. However, the underlying mechanism by which TDRD3 functions as a transcriptional coactivator is unknown. We identified topoisomerase IIIB (TOP3B) as a component of the TDRD3 complex. TDRD3 serves as a molecular bridge between TOP3B and arginine-methylated histones. The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter primarily by the H4R3me2a mark, and the complex promotes c-MYC gene expression. TOP3B relaxes negative supercoiled DNA and reduces transcription-generated R-loops in vitro. TDRD3 knockdown in cells increases R-loop formation at the c-MYC locus, and Tdrd3-null mice exhibit elevated R-loop formation at this locus in B cells. Tdrd3-null mice show significantly increased c-Myc/Igh translocation, a process driven by R-loop structures. By reducing negative supercoiling and resolving R-loop, TOP3B promotes transcription, protects against DNA damage and reduces the frequency of chromosomal translocations.
doi:10.1016/j.molcel.2014.01.011
PMCID: PMC3959860  PMID: 24507716
2.  Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1 
Molecular cell  2013;52(1):37-51.
Summary
The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.
doi:10.1016/j.molcel.2013.08.039
PMCID: PMC4129656  PMID: 24076217
3.  Wwox Hypomorphic Mice Display a Higher Incidence of B-Cell Lymphomas and Develop Testicular Atrophy 
Genes, chromosomes & cancer  2007;46(12):1129-1136.
WWOX is a putative tumor suppressor gene encoded within common chromosomal fragile site region FRA16D, in chromo-some band 16q23. Multiple studies have demonstrated that WWOX expression is often reduced or lost in various tumor types. WWOX tumor suppressor activity was suggested by re-expressing WWOX in breast, ovarian, and lung tumor cell lines leading to tumor growth inhibition in vivo. To determine whether loss of Wwox gene expression has a role in tumorigenesis, we generated a mouse strain containing a Wwox gene mutated by a gene-trap vector. Homozygous Wwox gene-trap mice (Wwoxgt/gt) had no detectable Wwox protein in most tissues examined, although, a low level could be detected in a minority of tissues. Because of these observations, we concluded that these mice are Wwox hypomorphs. Remarkably, Wwox hypomorphic mice are viable in contrast to the recently reported postnatal lethality of Wwox knockout mice. Testes from Wwoxgt/gt males had high numbers of atrophic seminiferous tubules and reduced fertility when compared with wild-type counterparts. We observed that the Wwoxgt/gt mice had a significantly shorter lifespan, and female hypomorphs had a higher incidence of spontaneous B-cell lymphomas. In conclusion, we describe a novel Wwox hypomorphic mouse model that overcomes postnatal lethality that was recently observed in Wwox knockout mice. Therefore, tumorigenesis studies using this model more closely recapitulates the loss of WWOX expression observed in human cancers. Importantly, our observation that Wwox hypomorphs had an increased incidence of B-cell lymphomas supports a role of Wwox as a tumor suppressor.
doi:10.1002/gcc.20497
PMCID: PMC4143238  PMID: 17823927
4.  WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins 
Oncogene  2004;23(29):5049-5055.
WWOX, the gene that maps to common chromosomal fragile site FRA16D, is frequently affected by aberrations in multiple types of cancers. WWOX encodes a 46 kDa protein that contains two WW domains and a short-chain oxidoreductase (SDR) domain. We recently demonstrated that ectopic expression of WWOX inhibits xenograft tumor growth of tumorigenic breast cancer cells. Little is known of the biochemical function(s) of WWOX. The SDR domain is predicted to be involved in sex-steroid metabolism and the WW domains are likely involved in protein–protein interactions. In this report, we identify the specific proline-rich ligand for WWOX as PPXY and show that the amino-terminal WW domain is responsible for this interaction. Using the WWOX WW domains as a probe, we screened high-density protein arrays and identified five candidate-binding partners. The binding to one of these candidates, small membrane protein of the lysosome/late endosome (SIMPLE), was further analysed, and we observed that a specific PPSY motif in the SIMPLE amino-acid sequence was required to interact with the amino-terminal WW domain of WWOX. In addition, immunofluorescence staining demonstrated that endogenous WWOX and SIMPLE co-localize to peri-nuclear compartments of MCF-7 human breast cancer cells. These studies demonstrate that WWOX contains a Group I WW domain that binds known cellular proteins containing the specific ligand PPXY. Identification and characterization of WWOX interacting proteins will lead to an understanding of the biological functions of WWOX in normal and tumor cells.
doi:10.1038/sj.onc.1207680
PMCID: PMC4143251  PMID: 15064722
tumor suppressors; WW domain; protein–protein interaction
5.  A gain-of-function mouse model identifies PRMT6 as a NF-κB coactivator 
Nucleic Acids Research  2014;42(13):8297-8309.
Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that modifies histone tails. To help elucidate the biological function of PRMT6 in vivo, we generated transgenic mice that ubiquitously express PRMT6 fused to the hormone-binding portion of the estrogen receptor (ER*). The ER*-PRMT6 fusion is unstable and cytoplasmic, but upon systemic treatment with tamoxifen, it becomes stabilized and translocates into the nucleus. As a result, a dramatic increase in the H3R2me2a histone mark is observed. We found that one consequence of induced ER*-PRMT6 activation is increased IL-6 levels. IL-6 expression is regulated by the nuclear factor-kappa B (NF-κB) transcription factor, and PRMT6 functions as a coactivator of this pathway. We show that PRMT6 directly interacts with RelA, and that its overexpression enhances the transcriptional activity of an ectopic NF-κB reporter and endogenously regulates NF-κB target genes. PRMT6 is recruited, by RelA, to selective NF-κB target promoters upon TNF-α stimulation. Moreover, ER*-PRMT6 activation causes RelA accumulation in the nucleus. In summary, we observe that PRMT6 is recruited to chromatin at selective NF-κB target promoters, where it likely impacts the histone code and/or methylates other chromatin-associated proteins to facilitate transcription.
doi:10.1093/nar/gku530
PMCID: PMC4117762  PMID: 24939901
6.  A TR-FRET based functional assay for screening activators of CARM1 
Chembiochem : a European journal of chemical biology  2013;14(7):10.1002/cbic.201300029.
Epigenome is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator Associated Arginine (R) Methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that overexpression of CARM1 by two-fold in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, leading to the hypothesis that activating CARM1 by chemical activators may be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved Förster resonance energy transfer (TR-FRET) assay using poly (A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen® TR-FRET assay utilizes MCF7 cells expressing GFP-PABP1 fusion protein via BacMam gene delivery system, methyl-PABP1 specific antibody and terbium-labeled secondary antibody. This assay has been validated to reflect the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.
doi:10.1002/cbic.201300029
PMCID: PMC3828750  PMID: 23585185
Arginine methylation; CARM1; PABP1; TR-FRET
8.  Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation* 
The Journal of Biological Chemistry  2014;289(12):8277-8287.
Background: SET7 monomethylates DNMT1, promoting its proteasomal degradation, yet methylated DNMT1 still remains throughout the cell cycle.
Results: The methyllysine reader PHF20L1 stabilizes methylated DNMT1. Disruption of PHF20L1 induces DNMT1 degradation and genome hypomethylation.
Conclusion: PHF20L1, an epigenetic reader, cooperates with writer and eraser to regulate epigenetic inheritance.
Significance: PHF20L1 can be targeted as a means of regulating DNMT1 activity and DNA methylation in cells.
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
doi:10.1074/jbc.M113.525279
PMCID: PMC3961655  PMID: 24492612
DNA-binding Protein; DNA Methylation; DNA Methyltransferase; Protein Degradation; Protein Methylation; MBT Domain; PHF20L1; SET7; UNC1215
9.  CARM1 regulates fetal hematopoiesis and thymocyte development 
CARM13 (coactivator-associated arginine methyltransferase 1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). Here we find that reduced thymopoiesis in Carm1−/− mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1−/− E14.5 or E18.5 fetal liver. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1−/− fetal liver cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1−/− fetal liver cells in a CARM1 sufficient host showed that CARM1 is required cell-autonomously in hematopoietic cells. Co-culture of Carm1−/− fetal liver cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.
doi:10.4049/jimmunol.1102513
PMCID: PMC3538901  PMID: 23248263
10.  Titivated for Destruction: the methyl-degron 
Molecular cell  2012;48(4):487-488.
In this issue, Lee et al. (2012) demonstrate that the degradation of RORα is regulated by the EZH2–DCAF1/DDB1/CUL4–proteasome axis, thus, identifying protein methylation as a post-translational modification that can orchestrate protein destruction through a motif termed the “methyl-degron.”
doi:10.1016/j.molcel.2012.11.007
PMCID: PMC3563253  PMID: 23200121
11.  Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1 
Journal of medicinal chemistry  2012;55(22):9875-9890.
Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators.
doi:10.1021/jm301097p
PMCID: PMC3508294  PMID: 23095008
CARM1 activator; PRMT inhibitors; arginine methyltransferase; histone modifying enzyme; epigenetics
12.  AMPK-Mediated Phosphorylation of Murine p27 at T197 Promotes Binding of 14-3-3 Proteins and Increases p27 Stability 
Molecular carcinogenesis  2010;49(5):10.1002/mc.20613.
The Tuberous Sclerosis Complex 2 (TSC2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability have not been elucidated. Previous studies have also shown that AMPK, which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198), which when AMPK is activated, results in stabilization of the p27 protein.
doi:10.1002/mc.20613
PMCID: PMC3818129  PMID: 20146253
Tuberous sclerosis complex; TSC2; energy sensing
13.  Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation 
Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.
doi:10.1074/mcp.O113.027870
PMCID: PMC3879628  PMID: 24129315
14.  Carm1 Regulates Pax7 Transcriptional Activity Through MLL1/2 Recruitment During Asymmetric Satellite Stem Cell Divisions 
Cell stem cell  2012;11(3):333-345.
SUMMARY
In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N-terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a novel reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.
doi:10.1016/j.stem.2012.07.001
PMCID: PMC3438319  PMID: 22863532
Satellite stem cells; Muscle regeneration; Pax7; Myf5; Carm1; Histone arginine methyltransferase; MLL; Histone H3K4 methyltransferase
15.  Discovery of a chemical probe for the L3MBTL3 methyl-lysine reader domain 
Nature chemical biology  2013;9(3):184-191.
We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
doi:10.1038/nchembio.1157
PMCID: PMC3577944  PMID: 23292653
16.  Association of UHRF1 with H3K9 methylation directs the maintenance of DNA methylation 
Nature structural & molecular biology  2012;19(11):1155-1160.
SUMMARY
A fundamental challenge in mammalian biology has been elucidating mechanisms linking DNA methylation and histone post-translational modifications. Human UHRF1 (ubiquitin-like, PHD and RING finger containing 1) has multiple domains that bind chromatin and is implicated genetically in DNA methylation maintenance. However, molecular mechanisms underlying DNA methylation regulation by UHRF1 are poorly defined. Here we show that UHRF1 association with methylated histone H3 lysine 9 (H3K9) is required for DNA methylation maintenance. We further show that UHRF1 association with H3K9 methylation is insensitive to adjacent H3 serine 10 phosphorylation – a known mitotic ‘phospho/methyl switch.’ Importantly, we demonstrate that UHRF1 mitotic chromatin association is necessary for DNA methylation maintenance through regulation of DNMT1 stability. Collectively, our results define a novel link between H3K9 methylation and the faithful epigenetic inheritance of DNA methylation, establishing an unexpected mitotic role for UHRF1 in this process.
doi:10.1038/nsmb.2391
PMCID: PMC3492551  PMID: 23022729
UHRF1; DNA methylation; histones; post-translational modifications; epigenetics
17.  PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53 
PHF20 is a multidomain protein and subunit of a lysine acetyltransferase complex that acetylates histone H4 and p53 but whose function is unclear. Using biochemical, biophysical and cellular approaches, we determined that PHF20 is a direct regulator of p53. A Tudor domain in PHF20 recognized p53 dimethylated at Lys370 or Lys382 and a homodimeric form of this Tudor domain could associate with the two dimethylated sites on p53 with enhanced affinity, indicating a multivalent interaction. Association with PHF20 promotes stabilization and activation of p53 by diminishing Mdm2-mediated p53 ubiquitylation and degradation. PHF20 contributes to upregulation of p53 in response to DNA damage, and ectopic expression of PHF20 in different cell lines leads to phenotypic changes that are hallmarks of p53 activation. Overall our work establishes that PHF20 functions as an effector of p53 methylation that stabilizes and activates p53.
doi:10.1038/nsmb.2353
PMCID: PMC3454513  PMID: 22864287
18.  Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs 
Scientific Reports  2013;3:1311.
Arginine methylation is a common posttranslational modification that is found on both histone and non-histone proteins. Three types of arginine methylation exist in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). PRMT1 is the primary methyltransferase that deposits the ADMA mark, and it accounts for over 90% of this type of methylation. Here, we show that with the loss of PRMT1 activity, there are major increases in global MMA and SDMA levels, as detected by type-specific antibodies. Amino acid analysis confirms that MMA and SDMA levels accumulate when ADMA levels are reduced. These findings reveal the dynamic interplay between different arginine methylation types in the cells, and that the pre-existence of the dominant ADMA mark can block the occurrence of SDMA and MMA marks on the same substrate. This study provides clear evidence of competition for different arginine methylation types on the same substrates.
doi:10.1038/srep01311
PMCID: PMC3575585  PMID: 23419748
19.  Novel 3,5-Bis(bromohydroxybenzylidene)piperidin-4-ones as Coactivator-associated Arginine Methyltransferase 1 Inhibitors: Enzyme Selectivity and Cellular Activity 
Journal of medicinal chemistry  2011;54(13):4928-4932.
Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis(3-bromo-4-hydroxybenzylidene) piperidin-4-one (7g) and its analogues 8a-l. Among them, 7g, 8e, and 8l displayed high and selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity.
doi:10.1021/jm200453n
PMCID: PMC3487391  PMID: 21612300
20.  Histone Arginine Methylation 
FEBS letters  2010;585(13):2024-2031.
Arginine methylation is a common posttranslational modification (PTM). This type of PTM occurs on both nuclear and cytoplasmic proteins, and is particularly abundant on shuttling proteins. In this review, we will focus on one aspect of this PTM: the diverse roles that arginine methylation of the core histone tails play in regulating chromatin function. A family of nine protein arginine methyltransferases (PRMTs) catalyze methylation reactions, and a subset target histones. Importantly, arginine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, thus playing a central role in the orchestration of the histone code.
doi:10.1016/j.febslet.2010.11.010
PMCID: PMC3409563  PMID: 21074527
arginine methylation; histone code; tudor domain; CARM1; ChIP-seq
21.  Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms 
The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function.
doi:10.1039/C0SC00297F
PMCID: PMC3399765  PMID: 22822423
22.  Discovery of Peptidylarginine Deiminase-4 Substrates by Protein Array: Antagonistic Citrullination and Methylation of Human Ribosomal Protein S2† 
Molecular bioSystems  2011;7(7):2286-2295.
Peptidylarginine deiminase (PAD) catalyzes the posttranslational citrullination of selected proteins in a calcium dependent manner. The PAD4 isoform has been implicated in multiple sclerosis, Rheumatoid arthritis, some types of cancer, and plays a role in gene regulation. However, the substrate selectivity of PAD4 is not well defined, nor is the impact of citrullination on many other pathways. Here, a high-density protein array is used as a primary screen to identify 40 previously unreported PAD4 substrates, 10 of which are selected and verified in a cell lysate-based secondary assay. One of the most prominent hits, human 40S ribosomal protein S2 (RPS2), is characterized in detail. PAD4 citrullinates the Arg-Gly repeat region of RPS2, which is also an established site for Arg methylation by protein arginine methyltransferase 3 (PRMT3). As in other systems, crosstalk is observed; citrullination and methylation modifications are found to be antagonistic to each other, suggesting a conserved posttranslational regulatory strategy. Both PAD4 and PRMT3 are found to co-sediment with the free 40S ribosomal subunit fraction from cell extracts. These findings are consistent with participation of citrullination in the regulation of RPS2 and ribosome assembly. This application of protein arrays to reveal new PAD4 substrates suggests a role for citrullination in a number of different cellular pathways.
doi:10.1039/c1mb05089c
PMCID: PMC3251905  PMID: 21584310
23.  Methylation by Set9 modulates FoxO3 stability and transcriptional activity 
Aging (Albany NY)  2012;4(7):462-479.
The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a ‘code’ affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the ‘histone code’. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.
PMCID: PMC3433933  PMID: 22820736
longevity; FOXO transcription factors; methyltransferase; Set9; aging
24.  Protein Arginine Methylation in Mammals: Who, What, and Why 
Molecular Cell  2009;33(1):1-13.
The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation.
doi:10.1016/j.molcel.2008.12.013
PMCID: PMC3372459  PMID: 19150423
25.  Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding 
Journal of molecular biology  2011;408(5):807-814.
M phase phosphoprotein 8 (MPP8) harbors a N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine 9 containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80, Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side chain hydroxyl oxygen of Tyr83 suggests the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).
doi:10.1016/j.jmb.2011.03.018
PMCID: PMC3081990  PMID: 21419134
X-ray crystallography; epigenetics; MPP8 phosphorylation; methyl-lysine binding

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